Carcinogenicity of dermally administered 1,2-dihydro-2,2,4-trimethylquinoline monomer in F344 rats and B6C3F1 mice.

1,2-Dihydro-2,2,4-trimethylquinoline (TMQ) was evaluated in a 2-year study in which groups of 60 male or female F344 rats received 0, 36 or 60 mg kg(-1) (0, 0.022, or 0.037 mg cm(-2)) and groups of 60 male or female B6C3F1 mice received 0, 3.6 or 10 mg kg(-1) (0, 0.00136, 0.00435 mg cm(-2)) in acetone by topical administration. Survival of all treated groups was comparable to survival of controls. Mean body weights of female rats were lower than those of controls throughout the study but mean body weights of male rats and male and female mice were comparable to the mean body weights of controls. No neoplasms of the skin were observed in any group of rats or mice. Acanthosis at the site of application was increased in male and female rats that received 60 or 100 mg kg(-1) and hyperkeratosis was increased in female rats that received 60 mg kg(-1). The incidences of renal tubule adenoma and renal tubule adenoma or carcinoma were increased significantly in the 60 and 100 mg kg(-1) groups of male rats. There were no neoplastic or non-neoplastic lesions in mice associated with exposure to 1,2-dihydro-2,2,4-trimethylquinoline. In a 1-year initiation-promotion study, groups of 30 female SENCAR mice received an initiating dose of 50 mg kg(-1) 1,2-dihydro-2,2,4-trimethylquinoline followed by promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA), or an initiating dose of 7,12-dimethylbenzanthracene (DMBA) followed by promotion with 5, 10 or 25 mg kg(-1) 1,2-dihydro-2,2,4-trimethylquinoline. Other groups served as initiator control, promoter control, vehicle control and positive control (DMBA initiation, TPA promotion). In this system, 1,2-dihydro-2,2,4-trimethylquinoline-initiated skin was not promoted by TPA, and DMBA-initiated skin was not promoted by 1,2-dihydro-2,2,4-trimethylquinoline.

Toxicity and carcinogenicity of delta 9-tetrahydrocannabinol in Fischer rats and B6C3F1 mice.

delta 9-Tetrahydrocannabinol (delta 9-THC) was studied for potential carcinogenicity in rodents because it is the principal psychoactive ingredient in marihuana and it has potential medicinal uses. delta 9-THC in corn oil was administered by gavage to groups of male and female Fischer rats and B6C3F1 mice at 0, 5, 15, 50, 150, or 500 mg/kg, 5 days a week for 13 weeks and for 13-week plus a 9-week recovery period, and to groups of rats at 0, 12.5, or 50 mg/kg and mice at 0, 125, 250, or 500 mg/kg, 5 times a week for 2 years. In all studies, mean body weights of dosed male and female rats and mice were lower than controls but feed consumptions were similar. Convulsions and hyperactivity were observed in dosed rats and mice; the onset and frequency were dose related. Serum FSH and LH levels in all dosed male rats and corticosterone levels in 25 mg/kg female rats were significantly higher than controls at 15 months in the 2-year studies. delta 9-THC administration for 13 weeks induced testicular atrophy and uterine and ovarian hypoplasia; the lesions persisted in a 9-week recovery period. In the 2-year studies, survival of dosed rats was higher than controls; that of mice was similar to controls. Incidences of testicular interstitial cell, pancreas and pituitary gland adenomas in male rats, mammary gland fibroadenoma and uterus stromal polyp in female rats, and hepatocellular adenoma/carcinoma in male and female mice were reduced in a dose-related manner. Decreased tumor incidences may be at least in part due to reduced body weights of dosed animals. Incidences of thyroid gland follicular cell hyperplasia were increased in all dosed groups of male and female mice, and follicular cell adenomas were significantly increased in the 125 mg/kg group of males, but there was no evidence of a dose-related trend in proliferative lesions of the thyroid. There was no evidence that delta 9-THC was carcinogenic in rats or mice.

Chemical effects in transgenic mice bearing oncogenes expressed in mammary tissue.

Three transgenic mouse lines carrying v-Ha-ras (TG-SH), c-myc (TG-M) or c-neu (TG-NK) oncogenes under regulatory control of mouse mammary tumor virus (MMTV) long terminal repeat (LTR) sequences were evaluated for responses to two chemical carcinogens. p-Cresidine, a mutagenic urinary bladder carcinogen, increased the incidence of urinary bladder carcinomas in males and females in all three lines, and these tumors occurred at comparable incidences and grade in transgenic and non-transgenic mice. p-Cresidine did not affect the rates of mammary or salivary gland neoplasms in transgenic mice; these tumors did not occur in non-transgenic littermates. No other tumor types were observed in exposed or control animals. Reserpine, a non-mutagenic mammary gland carcinogen, was administered under the same protocol, but the high control rates of mammary gland adenocarcinomas in the TG-M and TG-NK strains made it difficult to detect any tumor-enhancing effect of reserpine. However, the incidences of multiple mammary gland tumors were significantly increased in dosed females from both lines. The incidence of mammary gland adenocarcinomas was significantly increased in TG-SH females receiving 5 p.p.m. reserpine. Reserpine did not induce any carcinogenic effects in non-transgenic mice. These results indicate that the transcriptional regulation of these three transgenes is a major determinant in the response to p-cresidine and reserpine. The use of transgenic models for the general detection of carcinogens may require lines in which appropriate genes are targeted for expression in many tissues, or lines in which critical genes have been inactivated.

A high-yield sampler for toxicological characterization of complex mixtures in combustion effluents.

Combustion sampling for toxicological assessment often requires that large (greater than 100 mg) lots of complex organic mixtures of wide volatility range be rapidly recovered from high temperature gases without contamination. A new sampler, meeting these criteria for studies of public health interest, has been developed and demonstrated. The device provides high sampling rates and intimate contacting of the samples stream with large volumes of a well-cooled, liquid solvent, dichloromethane (DCM). This promotes rapid organics dissolution from carrier gas and particulates and prompt dilution and quenching of the resulting solution, resulting in high organics collection efficiencies with minimal DCM losses. Solvent separation then remits large quantities of concentrated organics for chemical analysis and toxicological testing. One- to seven-hour interrogations of in-flame, post-flame, and flue gas regions gave 50- to 250-mg yields of complex organic mixtures. In side-by-side sampling of combustion exhaust, the DCM sampler provided higher yields of DCM solubles (identified with complex organic mixtures) and of S. typhimuirim mutagens (active without exogenous metabolizing agents) than did a filter/polymeric sorbent bed sampling train. The new sampler also collects polar and high volatile hydrocarbons such as benzaheyde, pentadiyne, m- and p-diethynyl-benzene, and 1-hexen-3,5-diyne. Nitration of naphthalene and pyrene in DCM solution (1 mg/mL each) was less than 1 part in 10(7) after a 345-min exposure to a bubbling flow of moist N2/air mixture (1:1 v/v) containing 107 ppm NO and 1.5 ppm NO2, indicating that for these condition a DCM sampler should resist artifactual nitration of aromatics. However, because of the very high bacterial mutagenicity of some nitroaromatics and the wide range of sampling conditions of environmental interest, nitration and all artifacts must still be scrutinized when using the DCM sampler. The DCM sampler is expected to contribute to public health impact assessments by facilitating detailed determinations of the identities, compositions, concentrations, sources, formation mechanisms, and biological activity of environmental toxicants in gaseous atmospheres.

The relationship between mutagenicity and chemical composition of polycyclic aromatic compounds from coal pyrolysis.

The polycyclic aromatic compounds (PAC) produced from the pyrolysis of a bituminous coal at temperatures of 1125 to 1425 degrees K prove to be mutagenic to S. typhimurium, both in the presence and in the absence of postmitochondrial supernatant (PMS) prepared from Aroclor 1254-induced rat liver. Mutagenicity of the PAC samples measured in the absence of PMS exhibits little dependence on pyrolysis temperature; that measured in its presence is higher at the higher pyrolysis temperatures. However, because of the decrease in PAC yield as the temperature is raised, mutagenicity per mass of coal consumed falls with an increase in temperature if measured without PMS (-PMS) and peaks at an intermediate temperature of 1378 degrees K if measured with PMS (+PMS). Using a new chromatographic technique, we have split each coal-derived PAC sample into two fractions: LC1, containing PAC with alkyl and O-containing substitutions and LC2, consisting of unsubstituted PAC. Substituted (LC1) fractions show no significant +PMS mutagenicity, indicating that, as a whole, the alkylated PAC in our coal pyrolysis products are not mutagenic. Only at the higher temperatures do the substituted fractions exhibit significant -PMS mutagenicity, attributed to PAC with carbonyl or etheric functionalities. The extremely low yields of the substituted PAC under the conditions where they show some activity, however, ensure that they contribute little to overall mutagenicity. In contrast to the substituted fractions, the unsubstituted (LC2) fractions display significant mutagenicity under all conditions and appear to be responsible for virtually all of the mutagenicity in these coal-derived PAC samples. In this fraction, -PMS activity is attributed to nitrogen-containing heterocyclic aromatics.

The chemotherapeutic potential of glycol alkyl ethers: structure-activity studies of nine compounds in a Fischer-rat leukemia transplant model.

Structure-activity studies with nine glycol alkyl ethers were conducted with a cellular leukemia transplant model in male Fischer rats. This in vivo assay measures the effects of chemical treatment on neoplastic progression in transplant recipients. Chemicals were given ad libitum in the drinking water simultaneously with the transplants and continued throughout the study. In all, 20 million leukemic cells were injected s.c. into syngeneic rats, which after 60 days resulted in a 10-fold increase in relative spleen weights, a 100-fold increase in white blood cell counts, and a 50% reduction in red blood cell (RBC) indices and platelet counts. At this interval, ethylene glycol monomethyl ether (2-ME) given at a dose of 2.5 mg/ml in the drinking water completely eliminated all clinical, morphological, and histopathological evidence of leukemia, whereas the same dose of ethylene glycol monoethyl ether (2-EE) reduced these responses by about 50%. Seven of the glycol ethers were ineffective as anti-leukemic agents, including ethylene glycol, the monopropyl, monobutyl, and monophenyl ethylene glycol ethers, diethylene glycol, and the monomethyl and monoethyl diethylene glycol ethers. 2-ME more than doubled the latency period of leukemia expression and extended survival for at least 210 days. A minimal effective dose for a 50% reduction in the leukemic responses was 0.25 mg/ml 2-ME in the drinking water (15 mg/kg body weight), whereas a 10-fold higher dose of 2-EE was required for equivalent antileukemic activity. In addition, the in vitro exposure of a leukemic spleen mononuclear cell culture to 2-ME caused a dose- and time-dependent reduction in the number of leukemia cells after a single exposure to 1-100 microM concentrations, whereas the 2-ME metabolite, 2-methoxyacetic acid, was only half as effective. The two glycol alkyl ethers with demonstrable anti-leukemic activity, 2-ME and 2-EE, also exhibited a favorable efficacy-to-toxicity ratio and should be considered for further development as chemotherapeutic agents.

Chemical and toxicological characterization of residential oil burner emissions: II. Mutagenic, tumorigenic, and potential teratogenic activity.

Extracts of effluents from a modern residential oil burner have been evaluated in several toxicological assay systems. Bacterial mutagens were detected in extracts from both the particulate and vapor phase emissions. Effluents from continuous operation were an order of magnitude less mutagenic than those from cyclic (5 min on, 10 min off) operations. No difference in the yield of bacterial mutagens per gram of fuel burned was found between cyclic operation under low and moderate sooting conditions. On the basis of elution behavior from alumina it appeared that the bacterial mutagens collected from high sooting effluents were more polar than those from low sooting effluent. An extract that was mutagenic in bacteria did not induce a significant increase in mutation frequency to human lymphoblasts. No evidence of tumorigenicity was observed in a limited number of newborn mice after IP injection of effluent extract when compared to historical control data. Putative nonmutagenic teratogens were detected in effluent using an attachment inhibition assay. The level of these agents was reduced in effluents from continuous oil burner operation.

Organic emissions from coal pyrolysis: mutagenic effects.

Four different types of coal have been pyrolyzed in a laminar flow, drop tube furnace in order to establish a relationship between polycyclic aromatic compound (PAC) evolution and mutagenicity. Temperatures of 900K to 1700K and particle residence times up to 0.3 sec were chosen to best simulate conditions of rapid rate pyrolysis in pulverized (44-53 microns) coal combustion. The specific mutagenic activity (i.e., the activity per unit sample weight) of extracts from particulates and volatiles captured on XAD-2 resin varied with coal type according to the order: subbituminous greater than high volatile bituminous greater than lignite greater than anthracite. Total mutagenic activity (the activity per gram of coal pyrolyzed), however, varied with coal type according to the order: high volatile bituminous much greater than subbituminous = lignite much greater than anthracite, due primarily to high organic yield during high volatile bituminous coal pyrolysis. Specific mutagenic activity peaked in a temperature range of 1300K to 1500K and generally appeared at higher temperatures and longer residence times than peak PAC production.

Generation of biologically active substances in a natural gas flame.

Samples of gaseous and solid species taken from the central axis of a 1 megawatt heat-input natural gas flame were tested in vitro for mutagenic activity and teratogenic potential. Mutagenicity was determined by a Salmonella typhimurium forward mutation assay. Potential teratogenicity was indicated by the ability of samples to interfere with the attachment of mammalian cells to a lectin coated surface. Both the mutagenic and anti-attachment activities were found to peak in samples originating from the flame regions where the total polyaromatic compound (PAC) species concentration reached a maximum, indicating a strong correlation between PAC presence in the samples and biological activity. Additional anti-attachment activity was found close to the injection nozzle. No biologically active material was detected beyond the luminous portion of the flame.

Commercial hickory-smoke flavouring is a human lymphoblast mutagen but does not induce lung adenomas in newborn mice.

Commercial aqueous wood-smoke flavouring induced significant increases in the 6-thioguanine resistance mutation frequency of TK6 human lymphoblasts at 0.1 microliter flavouring/ml of cell suspension. This corresponds to 6 micrograms/ml of dissolved 'solids' as determined by fully drying the aqueous flavouring in a vacuum desiccator. In AHH-1 human lymphoblasts, which contain a cytochrome P-450 monooxygenase system, mutations were induced at 0.3 microliter/ml, corresponding to 18 microliters/ml of dissolved 'solids'. The flavouring did not induce 8-azaguanine resistant mutations in Salmonella typhimurium at concentrations up to 1.5 microliter/ml. At higher concentrations the flavouring was toxic to bacteria. The flavouring did not induce lung adenomas or other tumours in newborn mice when injected ip with total doses of up to 26 microliters over a 3-wk period. Toxicity to the kidney, colon and rectum was observed in some mice at 15 wk of age.

An internal survival standard for Salmonella typhimurium forward mutation assays.

A novel S. typhimurium forward mutation assay which avoids plating density artifacts is described. The new method uses a pair of multiply drug-resistant substrains of TM677, a bacterial strain used in previous forward mutation studies. This technique permits the measurement of cell survival following mutagen treatment by plating the culture on specially supplemented plates at the same cell concentration used to measure mutant yield. Thus this method is both technically easier and theoretically superior to our previous method.

Teratogen metabolism: thalidomide activation is mediated by cytochrome P-450.

A metabolite of thalidomide generated by hepatic microsomes inhibited the attachment of tumor cells to concanavalin A-coated polyethylene. Evidence that metabolite formation is mediated by microsomal cytochrome P-450 is presented. Microsomes incubated with thalidomide underwent a type I spectral shift. Metabolite formation was reduced or eliminated by carbon monoxide, SKF-525A, metyrapone, and N-octylamine. Superoxide dismutase treatment had no effect. Metabolite formation required microsomes and NADPH and was dependent on the length of 37 degrees C incubation. The metabolite could be isolated by successive hexane and chloroform extractions. It is likely the inhibitory thalidomide metabolite was generated by a minor cytochrome P-450 species. Whether this thalidomide metabolite is involved in the drug's teratogenic activity remains to be shown.

Teratogen metabolism: spontaneous decay products of thalidomide and thalidomide analogues are not bioactivated by liver microsomes.

Thalidomide and two analogues, EM87 and EM12, inhibited the attachment of tumor cells to concanavalin A-coated surfaces only if the drugs were first incubated with hepatic microsomes and cofactors. Most agents that inhibit attachment are demonstrated teratogens. Thalidomide undergoes spontaneous hydrolysis to at least 12 products in saline buffered to a pH of greater than 7. These hydrolysis products did not inhibit attachment nor could they be activated to inhibitory products with hepatic microsomes. Similarly EM12 and EM 87 hydrolysis products were neither inhibitory nor substrates for activation. If the three drugs were incubated in buffered saline, there was a progressive decline in their ability to act as substrates for activation to an inhibitory product. It was possible to remove microsomes from the incubation mixture following drug activation by centrifugation. This microsome-free mixture inhibited cell attachment. When mouse ovarian tumor (MOT) cells were added to the microsome-free mixture, attachment was inhibited. However, if the activated drugs were incubated in saline, there was a progressive decline in their ability to inhibit attachment. Decay rates differed for the three compounds. At a pH of 7.4, thalidomide, EM87, and EM12 required 3 h, 1h and 6h, respectively, to decay to control levels. These relative rates of decay are consistent with the relative teratogenicity of the three drugs.

Teratogen metabolism: activation of thalidomide and thalidomide analogues to products that inhibit the attachment of cells to concanavalin A coated plastic surfaces.

Thalidomide metabolites inhibited the attachment of tumor cells to concanavalin A coated polyethylene surfaces. Thalidomide, itself, was non-inhibitory. Thalidomide activation to inhibitory products required hepatic microsomes, an NADPH-generating system, and molecular oxygen. Production of inhibitory metabolites was unaffected by either epoxide hydrolase or 1,2-epoxy-3,3,3-trichloropropane (TCPO), an inhibitor of epoxide hydrolase endogenous to hepatic S9 fraction. Therefore, the attachment inhibitor was probably not an arene oxide. Inhibition was not accompanied by cytotoxicity, as judged by trypan blue exclusion. Although uninduced hepatic microsomes from mice, rats and dogs had similar abilities to activate thalidomide, microsomes from Aroclor 1254 induced rats were relatively inactive in the system. Inhibitory metabolites were generated from the thalidomide analogues EM8 , EM12 , EM16 , EM87 , EM136 , EM255 , E350 , phthalimide, phthalimido-phthalimide, indan, 1- indanone and 1,3- indandione . Glutarimide , glutamic acid and phthalic acid did not activate to inhibitory products.

Quantitative correspondence between the in vivo and in vitro activity of teratogenic agents.

We have tested 74 teratogenic and 28 nonteratogenic agents in a recently developed in vitro teratogen assay system. The assay identifies teratogens by their ability to inhibit attachment of ascites tumor cells to plastic surfaces coated with concanavalin A. There is a qualitative agreement between in vivo animal data and in vitro activity for 81 of the 102 agents (79%). Quantitative analysis shows a highly significant correlation coefficient of 0.69 between the inhibitory in vitro dose and the lowest reported teratogenic dose for 54 of the 60 inhibitory teratogens. The doses analyzed ranged over 5 orders of magnitude. We interpret these results to mean that attachment inhibition in concert with other, complementary, in vitro assay systems can become a useful method for the assessment of the teratogenic potential of environmental agents.

Inhibition of tumor cell attachment to concanavalin A-coated surfaces as an assay for teratogenic agents: approaches to validation.

Four environmental agents have been tested for activity in a recently developed in vitro teratogen assay system. All four agents inhibited attachment. The agents were 40-fold concentrated drinking water (ID50 = 0.45 ml/ml), whole cigarette smoke condensate (ID50 = 85 micrograms/ml), kerosene soot (ID50 = 90 micrograms/ml), and commercial formulations of the pesticide carbaryl (ID50 approximately 150 micrograms/ml). On the basis of these examples appropriate criteria for the validation of in vitro teratogen assay systems are discussed. It is concluded that criteria are critically dependent on the specific applications of the assay system. For example, the false-positive rate must be minimized to make a wide-ranging screen of water samples useful. On the other hand, an investigation of impurities in commercial compounds requires low false-negative rates. In every case a quantitative measure of the potential teratogenic potency, in vivo, is desirable.

Evidence of far ultraviolet light-mediated changes in plasma membrane structure and fuction.

Irradiation of plasma membrane preparations with 254 nm light increases its apparent microviscosity with fluorescent polarimetry. Doses of 3 . 10(4) J/m2 increase the fluorescent polarization of a diphenylhexatriene probe by 10%. A similar increase is seen when whole cells are irradiated. The fate of membrane protein following irradiation was examined using SDS-polyacrylamide gel electrophoresis. Increasing the 254 nm doses reduces the amount of material in distinct bands on the gel and increases the amount of very low mobility material. No new bands of Coomassie blue staining material were observed. Irradiation of whole cells inhibited their attachment to concanavalin A-coated surfaces, an indication of a change in membrane function.

Teratogenic drugs inhibit tumour cell attachment to lectin-coated surfaces.

Interactions between embryonic cells are generally thought to have a central role in the control of development. When these morphogenic interactions are interrupted by either physical intervention or genetic defects, normal development is impaired. In accord with these experiments, specific interactions between embryonic cells have been demonstrated in several in vitro systems. Many investigators have described homotypic aggregation of chick embryo cells, and heterotypic specificity has been described. Because of the importance of morphogenic cell-cell interactions in development it follows that agents that interfere with these interactions, regardless of the interference mechanism, are potential teratogens. Here we have used a simple in vitro cell to surface recognition system in an attempt to screen for potential teratogens. We have found a very high correlation between inhibitory activity in the in vitro assay and reported teratogenic activity in human or animal studies. This suggests that many teratogenic agents may act by interfering, in an as yet unknown way, in normal cell to cell interactions.